1. Field of the Invention
This invention relates to a fermentation method for the production of mannitol and to a mannitol dehydrogenase useful for producing mannitol from a variety of readily available carbohydrate substrates, especially fructose and glucose.
2. Description of the Prior Art
Mannitol, a naturally occurring polyol, is widely used in the food, pharmaceutical, medicine and chemical industries (Soetaert et al., Agro Food Ind. Hi-Tech. 6:41-44, 1995). It is used as a sweet-tasting bodying and texturing agent. Mannitol reduces the crystallization tendency of sugars and is used as such to increase the shelf-life of foodstuffs. Crystalline mannitol exhibits a very low hygroscopicity, making it useful in products that are stable at high humidity. It is extensively used in chewing gum. Because of its desirable properties, mannitol is commonly used in the pharmaceutical formulation of chewable tablets and granulated powders. It prevents moisture absorption from the air, exhibits excellent mechanical compressing properties, does not interact with the active components, and its sweet cool taste masks the unpleasant taste of many drugs (Debord et al., Drug Dev. Ind. Pharm. 13:1533-1546, 1987). The complex of boric acid with mannitol is used in the production of dry electrolytic capacitors. It is an extensively used polyol for production of resins and surfactants. Mannitol is used in medicine as a powerful osmotic diuretic and in many types of surgery for the prevention of kidney failure and to reduce dye and brain oedema. Mannitol hexanitrate is a well known vasodilator, used in the treatment of hypertension.
Mannitol is currently produced industrially by high pressure hydrogenation of fructose/glucose mixtures in aqueous solution at high temperature (120-160° C.) with Raney nickel as catalyst. Typically, the hydrogenation of a 50/50 fructose/glucose mixture results in an approximately 30/70 mixture of mannitol and sorbitol (Makkee et al., Starch/Starke 37:136-141, 1985). Therefore about half of the fructose is converted to mannitol and half of it to sorbitol. The glucose is hydrogenated exclusively to sorbitol. As a consequence, the commercial production of mannitol is always accompanied by the production of sorbitol, thus reducing the conversion efficiency of substrate to mannitol (Soetaert et al., 1995, supra).
In recent years, research efforts have been directed towards production of polyols by fermentation and enzymatic means (Vandamme et al. FEMS Microbiol. Rev. 16:163-186, 1995). Yun et al., (Biotechnol. Letts. 18:35-40, 1996) describe microbial transformation of fructose to mannitol by Lactobacillus sp. KY-107. In shake flask cultures, Yun et al. obtained a final concentration of 70 g mannitol/L from 100 g D-fructose within 80 h at 28° C. Yun et al. (J. Ferment. Bioeng. 85:203-208, 1998) report the isolation of two mannitol-producing, lactic acid bacteria from kimichi, a traditional Korean food. Lactobacillus sp. Y-107 transformed fructose to mannitol from the early growth stage, with a 54% conversion yield after 20 h; whereas Leuconostoc sp. Y-002 converted fructose to mannitol more slowly with a 40% yield at 20 h. Yun et al. (1998, supra) describe the pathway for microbial mannitol formation as comprising two mechanisms. In the first mechanism, NADPH-linked mannitol dehydrogenase directs the reduction of fructose. In the second mechanism, fructose-6-phosphate is initially reduced to mannitol-1-phosphate by means of NAD(P)H-linked mannitol-1-phosphate dehydrogenase. The mannitol-1-phosphate is then converted to inorganic phosphate and mannitol by means of a specific mannitol-1-phosphatase.
Korakli et al. (Adv. Food Sci. (CTML) 22:1-4, 2000) describe the production of mannitol in a fermentation process with selected strains of Lactobacillus sanfranciscensis with the ability to utilize maltose, sucrose and glucose as carbon sources. Cells of strain LTH 2590 were adapted to sucrose, but gave a decreased yield of mannitol production in relation to the fructose content of sucrose.
Itoh et al. (European Patent Number EP0486024, 1992) teaches the use of Lactobacillus sp. B001 (FERM BP-3158) for the production of mannitol, acetic acid and lactic acid on carbohydrate substrates comprising glucose and fructose. Itoh et al. reports obtaining a level of 12.3% mannitol in 23 h with a yield of sugar of 61%. Though being able of use other sugars, such as glucose, galactose, maltose and xylose, strain B001 does not metabolize mannose or trehalose.